INVITED REVIEW
Molecular
ROBERT J.
Biology in Nutrition
SMITH, MD
Harvard Medical School; Section on Metabolism, Joslin Brigham and Women’s Laboratory; New England Deaconess Hospital, Boston
ABSTRACT: Recent progress in the field of molecular
3.
biology has had a considerable effect on both basic and applied clinical nutrition and offers even greater promise for the future. It is important for individuals working in clinical
4.
nutrition,
as
well
as
basic science investigators, to have
Hospital; Joslin Clinic; and Research
appreciate the practical applications of molecular biology to the production of nutritionally important proteins. understand how nutrients may be used to modify specific gene expression.
an
understanding of the fundamental concepts of molecular biology in order to be able to evaluate and apply new approaches to clinical problems as they become available. The purpose of this review is to provide those who are not molecular biologists with a brief overview of the fundamental concepts of molecular biology and then to describe a number of the recent applications of molecular biological
Rapid developments in the field of molecular biology during the past 10 years have brought a revolution to the basic biological sciences and promise a revolution for the clinical sciences. During a very short period of time, a technology that was limited to specialized basic
methods and tools in the field of nutrition.
research laboratories has become accessible to and useful for basic and clinical researchers in many disciplines. Application of the science of molecular biology to practical clinical problems has yielded a growing list of &dquo;recombinant&dquo; human proteins, such as insulin,1 growth hormone,’ and insulin-like growth factor I (IGF-I),3 for use in disease therapy. The genetic bases of inherited disorders, such as Duchenne muscular dystrophy’ and cystic fibrosis,5 have been defined, and an enormous multicenter project aimed at the cloning and characterization of the entire human genome has been undertaken.’ In the field of nutrition, the technology of molecular biology has brought new products and analytical methods and will undoubtedly have a tremendous breadth of applications and impact. For those who are working in nutrition who are not molecular biologists, it will be important to acquire an understanding of the concepts of molecular biology in order to follow
LU CREDIT LEARNING OBJECTIVES-After reading &dquo;Molecular Biology in Nutrition&dquo; by Robert J. Smith, the reader will be able to: 1. understand some basic concepts regarding the normal flow of genetic information to the process of protein synthesis in the mammalian cell. 2. recognize some of the techniques that are used by the molecular biologist in studying nutritionally relevant metabolic processes.
Address for reprints: Robert J. Smith, MD, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215.
5
6 progress in biomedical research and to understand the implications of current research for present and future
clinical applications. This review will provide an introduction to the basic principles and terminology of molecular biology (see references 7 and 8 for more details). Important applications of specific molecular biological techniques in the field of nutrition will then be discussed. BASIC CONCEPTS
There is
an
incredible
diversity
and
complexity of
structures within the cells and tissues of humans and other organisms. These include major structures that are found in most cells, such as the nucleus, mito-
chondria, the Golgi apparatus, endoplasmic reticulum, and other organelles, and also specialized structures specific to certain differentiated cells, such as the myofibrillar contractile apparatus of skeletal muscle and the microvilli of intestinal absorptive cells. The structural complexity of mammalian cells has its basis in the regulated expression of thousands of different proteins. Most of the information that defines these molecules and structures is contained in DNA sequences within the cell nucleus. The primary focus of the technology of molecular biology is DNA and the processes that translate the informational content of DNA into cellular structures and functions. DNA is conceptually a very simple molecule, consisting of four different nucleic acids, thymine (T), cytosine (C), adenine (A), and guanine (G), linked together as nucleotide bases in long chains through a sugar phosphate backbone (Fig 1). Most of the time, DNA in the cell exists in a double-stranded form with T and A paired with each other and G and C paired with each other in the two strands. The specific A-T and G-C base pairing is the structural unit that holds the two strands together. Base pairing also provides a mechanism for generating new complementary strands from free unlinked nucleotides with a single strand of DNA as a template. Much of the informational content of DNA consists of regions of nucleotide or base sequences that define or code for the amino acid sequences of proteins. The steps involved in the information transfer from DNA to proteins is illustrated in Figure 2. The nucleotide sequence from defined regions of DNA is copied into a messenger RNA (mRNA) nucleotide sequence within the cell nucleus. This process is termed transcription. RNA has a slightly different sugar phosphate backbone but, like DNA, consists of a long strand of four nucleotides, guanine, cytosine, adenine, and uracil (present in place of the thymine of DNA). The sequence of the mRNA, or at least the part of the mRNA sequence contained in exons (discussed below), codes for the amino acid sequence of a specific protein. Because the formation of a protein on an mRNA template converts the language of nucleotides to the language of amino acids, with a triplet
Figure 1. Basic structure of DNA. Nucleotides are linked together within strands along a deoxyribose-phosphate backbone. Complementary strands are held together by base pairing between thymine (T) and adenine (A) or between cytosine (C) and guanine (G). of three nucleotides coding for each amino acid, the process has been termed translation. Ultimately, the proteins generated in this manner form cellular structures or have functions, for example as enzymes or membrane transporters, that dictate much of the overall structure and function of the cell. TRANSLATION AND NUTRITION
A closer review of the formation of proteins from mRNA illustrates an application of this knowledge in the field of nutrition. Protein synthesis occurs when specialized structures called ribosomes bind to one end of mRNA and move along the mRNA strand,
generating a progressively longer protein chain (Fig 3). After one ribosome moves out of the way, a second ribosome attaches, and the process continues until there may be many ribosomes attached to a single mRNA. These large structures, called polysomes, can be isolated by simple density gradient centrifugation techniques, and the total amount of RNA in polysomes
7
the third postoperative day. After the operation, there were significant decreases in total ribosome concentration and the percentage of ribosomes in polyribosomal structures in skeletal muscle with the control TPN formula. With the addition of glutamine to the TPN solution, the decreases in both total ribosomes and the percentage of polysomes were attenuated after the operation and did not differ significantly from preoperative values. The total content of polysomes was similar in the two groups preoperatively (Fig 4B, lanes 1 and 3) and appeared to decrease less in the skeletal muscle of patients given glutamine-containing as compared with glutamine-free TPN (lane 4 versus 2). This observation is of interest because it provides an in vivo index of protein synthesis in human muscle and it confirms the results of a number of animal studies that have shown anabolic effects of glutamine on muscle protein turnover. 12 The data support the conclusion that protein synthesis decreases postoperatively in both patient groups but that the decrease is blunted in patients maintained on glutamine-supplemented TPN. This evidence for a higher rate of protein synthesis in skeletal muscle with glutamine feeding offers at least a partial explanation (in addition to decreased protein degradation) for the improved nitrogen balance. cDNA CLONING
Although polysome analysis can be considered to be application of the tools of molecular biology in the field of nutrition, this methodology largely involves established biochemical techniques. The core of modern molecular biology, by contrast, employs novel methods based on the concept of cDNA cloning and an
Figure 2. Steps involved in information transfer from DNA protein. DNA is transcribed in the nucleus to an initial mRNA transcript. Introns are excised to generate a mRNA transcript containing only exon sequence. The final mRNA transcript is transferred out of the nucleus and is translated into protein.
to
the
use
of cDNA and related molecular constructs
as
experimental tools. An understanding of cDNA and its significance requires a consideration of mammalian genomic organization. It is known that DNA is not just an extensive sequence of millions of nucleotides strung together but rather that it is organized into distinct functional units
be measured.’ For a given tissue, the amount of RNA in polysomes is related to the overall rate of protein synthesis.10 Thus, it is possible to determine relative rates of protein synthesis under different conditions by comparing the quantity of polysomes within tissues. The practical application of this method is illustrated in a study by Hammarqvist et apl (Fig 4). These authors investigated the effects of adding the amino acid glutamine to a total parenteral nutrition (TPN) formula administered during the first 3 days after elective abdominal surgery for cholecystectomy. There was a significant improvement in nitrogen balance with the glutamine-containing TPN formula in comparison with the glutamine-free control formula (Fig 4A). In the same study patients, needle biopsies of skeletal muscle were obtained before operation and on can
totypical structural
designated as genes. A progene consists of the stretch of
DNA that encodes a specific protein and an adjacent stretch of regulatory DNA involved in determining when the gene is actively transcribed into mRNA and subsequently translated into protein (Fig 5). Cloning is the isolation and millionfold amplification of a stretch of DNA, such as this single gene, separate from all the rest of the cellular DNA so that it can be studied. The segment of DNA (or clone) of interest is cut away from the rest of the DNA by the use of enzymes purified from bacteria (restriction endonucleases) and is then inserted into a plasmid vector (Fig 6). The plasmid is a circular DNA molecule that generates millions of copies of itself when grown in the proper strain of bacteria. The plasmids can then be separated from the bacteria, the DNA clone can be cut back out
8
Figure 3. The structure of a polysome. Multiple ribosomes and partially synthesized (nascent) protein chains a single mRNA strand.
are
attached to
Figure 4. Effects of the addition of glutamine to TPN administered for 3 days after elective cholecystectomy. (A) Cumulative nitrogen balance. (B) Polysome content of quadriceps femoris muscle obtained by needle biopsy before and 3 days after the operation. Redrawn from data of Hammarqvist et ap1 with permission.
9
Figure
5.
Prototypical mammalian structural gene illustrating exons, introns, and a 5‘-untranslated regulatory region.
Figure 6. Basic principles of cDNA cloning. A chimeric plasmid is created by ligation of a fragment of cDNA into a plasmid vector (usually circular, double-stranded DNA) that has been opened by treatment with a restriction endonuclease. A large number of copies of the plasmid together with the inserted cDNA fragment can be generated by the infection of an appropriate bacterial strain.
with restriction enzymes, and millions of copies of the original DNA segment can thus be obtained for study. It is important to understand the concept of cDNA as compared with DNA. On further consideration of the prototypical mammalian structural gene with a
regulatory region and a structural region (Fig 5), it is evident that not all of the DNA sequence in the structural region gets transcribed into mRNA and translated into protein. Within the structural gene, there are blocks of DNA that code for protein sequence (designated exons) and intervening blocks of DNA that are cut out and eliminated during the processing of mRNA and, thus, are not present in the final mRNA sequence. These are designated introns. The essential features of mRNA processing are illustrated in Figure 2. First, an entire genomic DNA sequence is transcribed into an initial mRNA transcript (mRNA precursor). Then, the RNA undergoes processing or splicing reactions in which the introns are cut out and the exons are joined together, yielding a final mRNA transcript coding for the sequence ultimately represented in the corresponding protein. The presence of introns as well as exons for most mammalian genes has important influence on how we approach the problem of cloning. If the complete coding sequence of the final mRNA transcript is known, the sequence of its protein can be determined with a simple computer program that matches amino acids to the RNA triplet codons. If the complete sequence of the gene is known, however, and it is not known what is exon and what is intron, the protein sequence cannot be determined. For most experimental purposes, it would be desirable to know the mRNA sequence, but working directly with mRNA is difficult. In contrast to DNA, RNA is unstable and subject to degradation by RNases that are widespread in the environment and difficult to eliminate from experimental samples. For this reason, and for several other practical reasons, the process of cDNA cloning was developed. The steps involved in the generation of cDNA are described in Figure 7. In brief, mRNA is isolated and purified from a tissue of interest. The mRNA is then used as a template on which a strand of DNA complementary to the RNA sequence is formed (thus, the designation cDNA or complementary DNA). Most often, this is accomplished by using a short primer sequence of DNA that contains a string of thymine residues [oligo(dT) primer] and thus binds to the string of adenine residues [poly(A) tail] found in many mRNA. Starting at the end of this primer, the enzyme reverse transcriptase, which is isolated from DNA-dependent RNA viruses, is used to extend the complementary
10 sequence
along the
RNA strand. The
reverse
tran-
scriptase enzyme incorporates thymine residues rather than uracil, and thus, a double-stranded complex that contains one strand of DNA and one strand of RNA is generated. The strands are separated, the RNA is destroyed by alkali treatment, and the remaining DNA strand is used as a template to form a stable, doublestranded cDNA molecule that contains the sequence originally present in mRNA. It is beyond the scope of this article to provide the specific details, but many innovative methods have been developed for obtaining and isolating cDNA
clones
species
corresponding to individual proteins or mRNA of interest. Because cDNA is complementary
in sequence to mRNA and not to a gene sequence, there are no introns. The nucleotide sequence of the cDNA strand, which is relatively easy to obtain, can thus be used to deduce the sequence of the protein encoded by the original mRNA strand. Because it is stable and easily manipulated with many experimental tools, cDNA represents one of the most important cornerstones of modern molecular biology. ’
APPLICATIONS OF cDNA TECHNOLOGY
A listing of some of the practical applications of cDNA technology with relevance to the field of nutrition is presented in Table 1. The use of cDNA to derive the amino acid sequences of proteins was briefly noted above. Very rapid methods for obtaining the precise sequence of cDNA molecules have been developed which allow the analysis of hundreds of nucleotides per week in a small laboratory or thousands of nucleotides per week in a large specialized laboratory with automated procedures. Once the nucleotide sequence is known, the region of the cDNA that encodes its protein product can be viewed as a series of nucleotide triplets, with each unit of three nucleotides defining an amino acid in the protein (Fig 8). In this manner, it is possible to obtain information on protein structure that would otherwise require difficult and laborious biochemical purification procedures. Several examples of nutritionally relevant protein sequences derived from cDNA sequences are listed in Table 2. Sequence information obtained from the analysis of
7. Basic steps involved in the generation of cDNA from an mRNA template. A cDNA strand is generated with the enzyme reverse transcriptase, with a synthetic oligo-dT primer that attaches to the poly(A) tail of mRNA to initiate DNA synthesis. The RNA is degraded with alkali, and a second DNA strand is generated with the first strand as a template. Additional steps required for the processing of the DNA product are not illustrated.
Figure
Table 1. Practical
applications
Figure 8. Protein synthesis mechanism, illustrating the generation of a correct amino acid sequence by matching triplet codons with complementary sequences in amino acid-specific transfer RNA (tRNA) species.
of cDNA with relevance to nutrition
11
cDNA clones has contributed significantly to our knowledge of the structure, function, and regulation of each of these proteins, as well as their roles in human disease states. Northern Blotting. Once a cDNA clone has been obtained, it can be used as a probe to assess the level of expression of its corresponding mRNA in cells or tissues. This is most often accomplished by the method of Northern blotting, which is outlined in Figure 9. RNA is isolated from a tissue of interest and run through a gel matrix in an electrical field that spreads out or separates the different RNA species according to their sizes (ie, number of nucleotides). The RNA is then transferred from the gel to a paperlike material, usually nitrocellulose, by a process called blotting. The cDNA or a fragment of the cDNA corresponding to an mRNA of interest is made into a cDNA probe by radioactive labeling (usually with 32P)
and is allowed to interact with the blot. It attaches specifically to mRNA with complementary sequence through the process of base pairing. A piece of photographic film can then be placed over the blot, and the radiation released from the cDNA probe exposes the film. By this procedure, the location of a specific mRNA species on the blot (and thus its size) can be established and the amount of mRNA present can be determined from the density of the exposed region on the film. Figure 10 illustrates the use of the Northern blotting method to study the expression of mRNA for IGF-I in fetal liver during maternal fasting in the rat.13 Several IGF-I mRNA transcripts of different sizes are evident in the Northern blot of liver RNA from six control animals (shown in the upper panel of the figure). All of these mRNA species are markedly decreased in quantity in the livers of fetuses obtained from fasted pregnant rats (shown in the lower panel).
Table 2. Nutritionally relevant protein sequences derived from cDNA
Figure 9. Basic principles of Northern blotting. RNA is isolated from a tissue or cell of interest, separated on the basis of size by gel electrophoresis, and transferred to nitrocellulose paper. After allowing a labeled cDNA probe to hybridize to mRNA with a complementary nucleotide sequence, the specific mRNA species can be identified as individual bands by autoradiographically exposed film. The size of the mRNA can be determined by comparison with standards of known size, and the amount is proportional to the density of the band.
12
correctly translated by bacterial riboAlthough conceptually simple, the creation of protein-producing bacterial factories is a complex technology that has required: the construction of specialized plasmids called expression vectors, which produce a cDNA sequence readily translated into protein by bacteria, the use of bacterial strains optimized for protein production, and the application of innovative biochemical methods to finally obtain a protein that is close enough in structure to the human protein sequence
were
somes.
Figure 10. Northern blot illustrating the effects of maternal fasting on IGF-I mRNA in fetal rat liver. The multiple bands in each lane represent different-sized IGF-I mRNA species. Each numbered lane contains RNA from a different animal. Reproduced from Straus et aF3 with permission. kb, kilobase.
This hormone is an important determinant of tissue growth rates, and its regulation during states of nutritional deprivation is likely to be an important factor in determining fetal growth as well as associated congenital malformations. The Northern blotting technique can also be applied to human nutritional studies with RNA preparations obtained, for example, from needle biopsies of skeletal muscle.&dquo; Analysis of levels of mRNA coding for specific muscle proteins, such as myosin, can provide insight into the factors that influence protein synthetic rates and overall tissue nitrogen balance. Recombinant Protein Products from Bacteria. The power of cDNA technology is dependent in large part on the availability of relatively simple methods to greatly amplify the number of copies of a specific cDNA sequence once it has been cloned. As was illustrated in Figure 6, this can be accomplished simply by infecting a culture of bacteria with a plasmid that contains the cDNA of interest and then allowing the plasmid to replicate to high levels within the bacteria. The host bacteria become cDNA-producing biological factories. Because the cDNA contains a sequence that is equivalent to the nucleotide sequence of mRNA, large quantities of the protein encoded by the cDNA could be synthesized by the bacteria if the cDNA
of interest to be useful. The economic incentives and the potential clinical importance of producing large quantities of human proteins through these recombinant DNA methods have stimulated an enormous amount of research and have led to considerable success in this area in the past several years. Table 3 presents a partial listing of proteins with importance in the field of nutrition that have been produced by this application of recombinant DNA1 technology. The use of human recombinant insulin has largely replaced the use of pork and beef insulin for the treatment of diabetes mellitus in many parts of the world and has averted a potential crisis as the need for insulin threatened to exceed available supplies from slaughterhouse-derived animal tissues.&dquo; Human recombinant growth hormone’ became available for clinical use in children with growth hormone deficiency at about the time that human pituitaryderived growth hormone was recognized to be hazardous because of potential pathological viral contaminants. 16 Recent studies have suggested that growth hormone may have therapeutic usefulness as an anabolic agent in nutritionally depleted patients. 17 Other hormones, such as IGF-I18 and granulocyte-macrophage colony stimulating factor,19 may also have important clinical applications. The recognition that trophic hormones and nutrients may have additive anabolic effects or may even act synergistically2° has generated a considerable amount of interest among nutrition researchers. A new era in the field of nutritional support may develop in which the specific composition and quantity of nutrients provided to many catabolic, critically ill patients are determined, in part, by the coordinate administration of recombinant, tissue-specific trophic hormones.
Recombinant Protein Expression in Cultured Cells. By using methods conceptually similar to those that Table 3. Nutritionally relevant recombinant DNA technology
proteins produced by
13
have been used to introduce recombinant DNA into bacteria and to induce the production of specific cloned proteins, it is possible to modify mammalian cells in culture such that they also produce large quantities of proteins encoded by cDNA clones.21 The introduction of foreign cDNA into mammalian cells, through the process called transfection, provides a powerful tool for studying the function of both normal and abnormal proteins. Proteins involved in nutritionally important pathways, such as insulin receptors,22 IGF receptors,23 or low-density lipoprotein receptors,24 can be transfected into cultured cells, and the consequences of their expression at high levels on the cell surface can be studied to gain insight into their normal effects on cellular metabolism. By modifying the sequence of the transfected cDNA by methods of sitedirected mutagenesis,25 the structures of the expressed proteins can be changed and the specific relationships between protein structure and function can be defined.26 Naturally occurring mutant cDNA sequences isolated from patients with inherited metabolic disorders can similarly be transfected into cultured cells, and the consequences of the mutation for the function of the protein can be investigated. By this method, it has recently been possible to demonstrate the molecular basis for certain rare forms of diabetes and growth disorders.&dquo; As the genetic bases for inherited propensities to many nutritionally related disorders, including various forms of obesity, lipid disorders, and digestive disorders, are identified in the years ahead, studies in transfected cultured cells will provide a valuable tool for establishing the functional properties of the abnormal or modified proteins and, thus, the basic molecular events that result in clinical disease. Gene and Protein Expression in Intact Organisms. An exciting recent application of transfection techniques is their use to introduce cDNA into intact animals. An animal that has received foreign DNA in this manner is referred to as transgenic.28 This is an area of intense research interest, because it offers the potential not only to investigate protein functions in vivo in a manner analogous to that described for transfected cultured cells, but it also has possible therapeutic applications as one form of corrective gene therapy. Most work has thus far been carried out in mice, and the method most commonly employed at present involves the injection of a specialized plasmid containing the cDNA of interest into a fertilized mouse egg under direct vision (Fig 11). Several eggs are then implanted into a hormonally primed female mouse, and the progeny are examined for the presence of the injected DNA sequence and phenotypic or biochemical consequences of the expression of the protein encoded by the injected DNA sequence. Under favorable conditions, the injected DNA is stably incorporated into the mouse genome and thus becomes an inherited trait that can be studied in the progeny of the initial transgenic mouse. The result can be dra-
11. Microinjection of fertilized mouse egg with foreign DNA. The egg is immobilized on a pipet by gentle suction. Reproduced from Gordon and Ruddle29 with permission.
Figure
matic, as illustrated in the transgenic mouse in Figure 12 expressing high levels of an injected growth hormone
gene.3° This methodology has obvious important
implications for studying protein functions in vivo and for correcting genetic abnormalities. It also has potential applications as a method for modifying the quality and composition of natural nutritional products obtained from plant and animal sources. It has become near-future science fiction to envision &dquo;transgenic supercattle&dquo; producing low-fat milk, low-cholesterol beef, or even human milk.31 SUMMARY AND A LOOK TO THE FUTURE
In the past several years, there has been a flourishing of new technologies as the science of molecular biology has rapidly developed. It has been possible in this review to discuss briefly only a few of the methods and experimental approaches that have current or potential application in the field of nutrition. Some of the applications of this technology that can be expected in the near future are summarized in Table 4. The cloning of cDNA corresponding to enzymes, transport proteins, hormones, plasma proteins, and other diverse proteins that have nutritionally relevant functions can be anticipated. By methods similar to those described in this review, the study of these proteins will yield new insights into molecular regulatory mechanisms and will also lead to the development of new recombinant DNA products. By powerful new methods for studying the genetic basis of disease at the molecular level, the specific genetic determinants of disease and risk factors for diseases such as obesity, atherosclerosis, neoplasia, and classical inborn errors of metabolism will be defined at an even more rapid pace. Methods are being developed for altering these genetic factors through gene modification or various forms of gene replacement. New disciplines may develop within the field of nutrition that are defined by this new knowledge. For example, as
14 REFERENCES 1. Villa-Komaroff L, Efstratiadis A, Broome S, et al. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci. USA 1978;
75:3727-31. 2. Martial
3.
4.
5. 6. 7. 8.
9.
Figure
12. Two
sibling male
mice at approximately 10 weeks of age. The animal on the left (44 g) contains the rat growth hormone gene fused to the mouse metallothionein promoter. The animal on the right (29 g) is a normal control. Photograph courtesy of Ralph Brinster.
Table 4. Future in nutrition
10.
11.
applications of molecular biology
JA, Hallewell RA, Baxter JD, et al. Human growth hormone: complementary DNA. Cloning and expression in bacteria. Science 1979;205:602-7. Buell G, Schulz MF, Selzer G, et al. Optimizing the expression of a synthetic gene encoding somatomedin-C (IGF-I). Nucleic Acids Res 1985;13:1923-38. Kunkel LM, Hoffman EP. Duchenne/Becker muscular dystrophy : a short overview of the gene, the protein; and current diagnostics. Br Med Bull 1989;45:630-43. McPherson MA, Dormer RL. Molecular and cellular biology of cystic fibrosis. Mol Aspects Med 1991;12:1-81. Allende JE. The human genome initiative. FASEB J 1991;5: 6-78. Lewin B. Genes IV. Cambridge: Cell Press, 1990. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989. Wernerman J, von der Decken A, Vinnars E. Size distribution of ribosomes in biopsy specimens of human skeletal muscle during starvation. Metabolism 1985;34:665-9. Wernerman J, von der Decken A, Vinnars E. The interpretation of ribosome determination to assess protein synthesis in human skeletal muscle. Infusionstherapie 1986;13:162-5. Hammarqvist F, Wernerman J, Ali R, et al. Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis, and improves nitrogen balance. Ann
Surg 1989;209:455-61. RJ, Wilmore DW. Glutamine nutrition and require1990;14:94S-9S. 13. Straus DS, Ooi GT, Orlowski CC, et al. Expression of the genes for insulin-like growth factor-I (IGF-I), IGF-II, and IGF-bind-
12. Smith
ments. JPEN
ing proteins-1 and -2 in fetal rat under conditions of intrauterine growth retardation caused by maternal fasting. Endocrinol14.
15.
ogy 1991;128:518-25. Fong Y, Minei JP, Marano MA, et al. Skeletal muscle amino acid and myofibrillar protein mRNA response to thermal injury and infection. Am J Physiol 1991;261:R536-42. Brogden RN, Heel RC. Human insulin. A review of its biological activity, pharmacokinetics and therapeutic use. Drugs 1987;34:
350-71. 16. Tintner
of the regulation of the expression of specific genes advances, there may be opportunities for directed nutrient regulation of target genes as a component of practical therapy. It should be apparent to the reader that molecular biology can be viewed as a set of new tools available to basic and clinical researchers. To be effectively used, this methodology must ultimately be integrated with other basic sciences, such as biochemistry, metabolism, and physiology, as well as with the clinical disciplines that directly confront the problems presented by human disease. The challenge for health care workers in clinical nutrition is to understand the new technology and to actively participate in its Ma effective application to clinical practice.
knowledge
m
17.
18.
19.
20.
21.
R, Brown P, Hedley-Whyte ET, et al. Neuropathologic
verification of Creutzfeldt-Jakob disease in the exhumed American recipient of human pituitary growth hormone: epidemiologic and pathogenetic implications. Neurology 1986;36:932-6. Manson JM, Smith RJ, Wilmore DW. Growth hormone stimulates protein synthesis during hypocaloric parenteral nutrition. Role of hormonal-substrate environment. Ann Surg 1988; 208:136-42. Skottner A, Clark RG, Fryklund L, et al. Growth responses in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor I. Endocrinology 1989;124: 2519-26. Crawford J, Ozer H, Stoller R, et al. Reduction by granulocyte colony-stimulating factor of fever and neutropenia induced by chemotherapy in patients with small-cell lung cancer. N Engl J Med 1991;325:164-70. Jacobs DO, Evans DA, Mealy K, et al. Combined effects of glutamine and epidermal growth factor on the rat intestine.
Surgery 1988;104:358-64. Scangos G, Ruddle FH. Mechanisms and applications of DNA mediated gene transfer in mammalian cells; a review. Gene 1981;14:1-10.
ACKNOWLEDGMENT
The author thanks Drs. Michael Pedrini and Thomas R. Ziegler for careful reading of the manu-
script.
22. Ebina Y, Edery M, Ellis L, et al. Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells. Proc Natl Acad Sci USA 1985;82:8014-8. 23. Steele-Perkins G, Turner J, Edman JC, et al. Expression and characterization of a functional human insulin-like growth factor I receptor. J Biol Chem 1988;263:11486-92.
15 24.
Miyanohara A, Sharkey MF, Witztum JL, et al. Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit
fibroblasts in vitro. Proc Natl Acad Sci USA 1988;85:6538-42. 25. Harris T. In vitro mutagenesis. Nature 1982;299:298-9. 26. Ellis L, Clauser E, Morgan DO. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell 1986; 45:721-32. 27. Moller DE, Flier JS. Mechanisms of disease. Insulin resistance—mechanisms, syndromes, and implications. N Engl J
Med 1991;325:938-48. RD, Brinster RL. Germ-line transformation of mice. Annu Rev Genet 1986;20:465-99. 29. Gordon JW, Ruddle FH. Gene transfer into mouse embryos: production of transgenic mice by pronuclear injection. Methods
28. Palmiter
Enzymol 1982;101:411-33. 30. Palmiter RD, Brinster RL, Hammer RE, et al. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 1982;300:611-5. 31. Johnson J. Transgenics: the land of milk and money. J NIH Res 1991;3:26-7.
Molecular
ROBERT J.
Biology in Nutrition
SMITH, MD
Harvard Medical School; Section on Metabolism, Joslin Brigham and Women’s Laboratory; New England Deaconess Hospital, Boston
ABSTRACT: Recent progress in the field of molecular
3.
biology has had a considerable effect on both basic and applied clinical nutrition and offers even greater promise for the future. It is important for individuals working in clinical
4.
nutrition,
as
well
as
basic science investigators, to have
Hospital; Joslin Clinic; and Research
appreciate the practical applications of molecular biology to the production of nutritionally important proteins. understand how nutrients may be used to modify specific gene expression.
an
understanding of the fundamental concepts of molecular biology in order to be able to evaluate and apply new approaches to clinical problems as they become available. The purpose of this review is to provide those who are not molecular biologists with a brief overview of the fundamental concepts of molecular biology and then to describe a number of the recent applications of molecular biological
Rapid developments in the field of molecular biology during the past 10 years have brought a revolution to the basic biological sciences and promise a revolution for the clinical sciences. During a very short period of time, a technology that was limited to specialized basic
methods and tools in the field of nutrition.
research laboratories has become accessible to and useful for basic and clinical researchers in many disciplines. Application of the science of molecular biology to practical clinical problems has yielded a growing list of &dquo;recombinant&dquo; human proteins, such as insulin,1 growth hormone,’ and insulin-like growth factor I (IGF-I),3 for use in disease therapy. The genetic bases of inherited disorders, such as Duchenne muscular dystrophy’ and cystic fibrosis,5 have been defined, and an enormous multicenter project aimed at the cloning and characterization of the entire human genome has been undertaken.’ In the field of nutrition, the technology of molecular biology has brought new products and analytical methods and will undoubtedly have a tremendous breadth of applications and impact. For those who are working in nutrition who are not molecular biologists, it will be important to acquire an understanding of the concepts of molecular biology in order to follow
LU CREDIT LEARNING OBJECTIVES-After reading &dquo;Molecular Biology in Nutrition&dquo; by Robert J. Smith, the reader will be able to: 1. understand some basic concepts regarding the normal flow of genetic information to the process of protein synthesis in the mammalian cell. 2. recognize some of the techniques that are used by the molecular biologist in studying nutritionally relevant metabolic processes.
Address for reprints: Robert J. Smith, MD, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215.
5
6 progress in biomedical research and to understand the implications of current research for present and future
clinical applications. This review will provide an introduction to the basic principles and terminology of molecular biology (see references 7 and 8 for more details). Important applications of specific molecular biological techniques in the field of nutrition will then be discussed. BASIC CONCEPTS
There is
an
incredible
diversity
and
complexity of
structures within the cells and tissues of humans and other organisms. These include major structures that are found in most cells, such as the nucleus, mito-
chondria, the Golgi apparatus, endoplasmic reticulum, and other organelles, and also specialized structures specific to certain differentiated cells, such as the myofibrillar contractile apparatus of skeletal muscle and the microvilli of intestinal absorptive cells. The structural complexity of mammalian cells has its basis in the regulated expression of thousands of different proteins. Most of the information that defines these molecules and structures is contained in DNA sequences within the cell nucleus. The primary focus of the technology of molecular biology is DNA and the processes that translate the informational content of DNA into cellular structures and functions. DNA is conceptually a very simple molecule, consisting of four different nucleic acids, thymine (T), cytosine (C), adenine (A), and guanine (G), linked together as nucleotide bases in long chains through a sugar phosphate backbone (Fig 1). Most of the time, DNA in the cell exists in a double-stranded form with T and A paired with each other and G and C paired with each other in the two strands. The specific A-T and G-C base pairing is the structural unit that holds the two strands together. Base pairing also provides a mechanism for generating new complementary strands from free unlinked nucleotides with a single strand of DNA as a template. Much of the informational content of DNA consists of regions of nucleotide or base sequences that define or code for the amino acid sequences of proteins. The steps involved in the information transfer from DNA to proteins is illustrated in Figure 2. The nucleotide sequence from defined regions of DNA is copied into a messenger RNA (mRNA) nucleotide sequence within the cell nucleus. This process is termed transcription. RNA has a slightly different sugar phosphate backbone but, like DNA, consists of a long strand of four nucleotides, guanine, cytosine, adenine, and uracil (present in place of the thymine of DNA). The sequence of the mRNA, or at least the part of the mRNA sequence contained in exons (discussed below), codes for the amino acid sequence of a specific protein. Because the formation of a protein on an mRNA template converts the language of nucleotides to the language of amino acids, with a triplet
Figure 1. Basic structure of DNA. Nucleotides are linked together within strands along a deoxyribose-phosphate backbone. Complementary strands are held together by base pairing between thymine (T) and adenine (A) or between cytosine (C) and guanine (G). of three nucleotides coding for each amino acid, the process has been termed translation. Ultimately, the proteins generated in this manner form cellular structures or have functions, for example as enzymes or membrane transporters, that dictate much of the overall structure and function of the cell. TRANSLATION AND NUTRITION
A closer review of the formation of proteins from mRNA illustrates an application of this knowledge in the field of nutrition. Protein synthesis occurs when specialized structures called ribosomes bind to one end of mRNA and move along the mRNA strand,
generating a progressively longer protein chain (Fig 3). After one ribosome moves out of the way, a second ribosome attaches, and the process continues until there may be many ribosomes attached to a single mRNA. These large structures, called polysomes, can be isolated by simple density gradient centrifugation techniques, and the total amount of RNA in polysomes
7
the third postoperative day. After the operation, there were significant decreases in total ribosome concentration and the percentage of ribosomes in polyribosomal structures in skeletal muscle with the control TPN formula. With the addition of glutamine to the TPN solution, the decreases in both total ribosomes and the percentage of polysomes were attenuated after the operation and did not differ significantly from preoperative values. The total content of polysomes was similar in the two groups preoperatively (Fig 4B, lanes 1 and 3) and appeared to decrease less in the skeletal muscle of patients given glutamine-containing as compared with glutamine-free TPN (lane 4 versus 2). This observation is of interest because it provides an in vivo index of protein synthesis in human muscle and it confirms the results of a number of animal studies that have shown anabolic effects of glutamine on muscle protein turnover. 12 The data support the conclusion that protein synthesis decreases postoperatively in both patient groups but that the decrease is blunted in patients maintained on glutamine-supplemented TPN. This evidence for a higher rate of protein synthesis in skeletal muscle with glutamine feeding offers at least a partial explanation (in addition to decreased protein degradation) for the improved nitrogen balance. cDNA CLONING
Although polysome analysis can be considered to be application of the tools of molecular biology in the field of nutrition, this methodology largely involves established biochemical techniques. The core of modern molecular biology, by contrast, employs novel methods based on the concept of cDNA cloning and an
Figure 2. Steps involved in information transfer from DNA protein. DNA is transcribed in the nucleus to an initial mRNA transcript. Introns are excised to generate a mRNA transcript containing only exon sequence. The final mRNA transcript is transferred out of the nucleus and is translated into protein.
to
the
use
of cDNA and related molecular constructs
as
experimental tools. An understanding of cDNA and its significance requires a consideration of mammalian genomic organization. It is known that DNA is not just an extensive sequence of millions of nucleotides strung together but rather that it is organized into distinct functional units
be measured.’ For a given tissue, the amount of RNA in polysomes is related to the overall rate of protein synthesis.10 Thus, it is possible to determine relative rates of protein synthesis under different conditions by comparing the quantity of polysomes within tissues. The practical application of this method is illustrated in a study by Hammarqvist et apl (Fig 4). These authors investigated the effects of adding the amino acid glutamine to a total parenteral nutrition (TPN) formula administered during the first 3 days after elective abdominal surgery for cholecystectomy. There was a significant improvement in nitrogen balance with the glutamine-containing TPN formula in comparison with the glutamine-free control formula (Fig 4A). In the same study patients, needle biopsies of skeletal muscle were obtained before operation and on can
totypical structural
designated as genes. A progene consists of the stretch of
DNA that encodes a specific protein and an adjacent stretch of regulatory DNA involved in determining when the gene is actively transcribed into mRNA and subsequently translated into protein (Fig 5). Cloning is the isolation and millionfold amplification of a stretch of DNA, such as this single gene, separate from all the rest of the cellular DNA so that it can be studied. The segment of DNA (or clone) of interest is cut away from the rest of the DNA by the use of enzymes purified from bacteria (restriction endonucleases) and is then inserted into a plasmid vector (Fig 6). The plasmid is a circular DNA molecule that generates millions of copies of itself when grown in the proper strain of bacteria. The plasmids can then be separated from the bacteria, the DNA clone can be cut back out
8
Figure 3. The structure of a polysome. Multiple ribosomes and partially synthesized (nascent) protein chains a single mRNA strand.
are
attached to
Figure 4. Effects of the addition of glutamine to TPN administered for 3 days after elective cholecystectomy. (A) Cumulative nitrogen balance. (B) Polysome content of quadriceps femoris muscle obtained by needle biopsy before and 3 days after the operation. Redrawn from data of Hammarqvist et ap1 with permission.
9
Figure
5.
Prototypical mammalian structural gene illustrating exons, introns, and a 5‘-untranslated regulatory region.
Figure 6. Basic principles of cDNA cloning. A chimeric plasmid is created by ligation of a fragment of cDNA into a plasmid vector (usually circular, double-stranded DNA) that has been opened by treatment with a restriction endonuclease. A large number of copies of the plasmid together with the inserted cDNA fragment can be generated by the infection of an appropriate bacterial strain.
with restriction enzymes, and millions of copies of the original DNA segment can thus be obtained for study. It is important to understand the concept of cDNA as compared with DNA. On further consideration of the prototypical mammalian structural gene with a
regulatory region and a structural region (Fig 5), it is evident that not all of the DNA sequence in the structural region gets transcribed into mRNA and translated into protein. Within the structural gene, there are blocks of DNA that code for protein sequence (designated exons) and intervening blocks of DNA that are cut out and eliminated during the processing of mRNA and, thus, are not present in the final mRNA sequence. These are designated introns. The essential features of mRNA processing are illustrated in Figure 2. First, an entire genomic DNA sequence is transcribed into an initial mRNA transcript (mRNA precursor). Then, the RNA undergoes processing or splicing reactions in which the introns are cut out and the exons are joined together, yielding a final mRNA transcript coding for the sequence ultimately represented in the corresponding protein. The presence of introns as well as exons for most mammalian genes has important influence on how we approach the problem of cloning. If the complete coding sequence of the final mRNA transcript is known, the sequence of its protein can be determined with a simple computer program that matches amino acids to the RNA triplet codons. If the complete sequence of the gene is known, however, and it is not known what is exon and what is intron, the protein sequence cannot be determined. For most experimental purposes, it would be desirable to know the mRNA sequence, but working directly with mRNA is difficult. In contrast to DNA, RNA is unstable and subject to degradation by RNases that are widespread in the environment and difficult to eliminate from experimental samples. For this reason, and for several other practical reasons, the process of cDNA cloning was developed. The steps involved in the generation of cDNA are described in Figure 7. In brief, mRNA is isolated and purified from a tissue of interest. The mRNA is then used as a template on which a strand of DNA complementary to the RNA sequence is formed (thus, the designation cDNA or complementary DNA). Most often, this is accomplished by using a short primer sequence of DNA that contains a string of thymine residues [oligo(dT) primer] and thus binds to the string of adenine residues [poly(A) tail] found in many mRNA. Starting at the end of this primer, the enzyme reverse transcriptase, which is isolated from DNA-dependent RNA viruses, is used to extend the complementary
10 sequence
along the
RNA strand. The
reverse
tran-
scriptase enzyme incorporates thymine residues rather than uracil, and thus, a double-stranded complex that contains one strand of DNA and one strand of RNA is generated. The strands are separated, the RNA is destroyed by alkali treatment, and the remaining DNA strand is used as a template to form a stable, doublestranded cDNA molecule that contains the sequence originally present in mRNA. It is beyond the scope of this article to provide the specific details, but many innovative methods have been developed for obtaining and isolating cDNA
clones
species
corresponding to individual proteins or mRNA of interest. Because cDNA is complementary
in sequence to mRNA and not to a gene sequence, there are no introns. The nucleotide sequence of the cDNA strand, which is relatively easy to obtain, can thus be used to deduce the sequence of the protein encoded by the original mRNA strand. Because it is stable and easily manipulated with many experimental tools, cDNA represents one of the most important cornerstones of modern molecular biology. ’
APPLICATIONS OF cDNA TECHNOLOGY
A listing of some of the practical applications of cDNA technology with relevance to the field of nutrition is presented in Table 1. The use of cDNA to derive the amino acid sequences of proteins was briefly noted above. Very rapid methods for obtaining the precise sequence of cDNA molecules have been developed which allow the analysis of hundreds of nucleotides per week in a small laboratory or thousands of nucleotides per week in a large specialized laboratory with automated procedures. Once the nucleotide sequence is known, the region of the cDNA that encodes its protein product can be viewed as a series of nucleotide triplets, with each unit of three nucleotides defining an amino acid in the protein (Fig 8). In this manner, it is possible to obtain information on protein structure that would otherwise require difficult and laborious biochemical purification procedures. Several examples of nutritionally relevant protein sequences derived from cDNA sequences are listed in Table 2. Sequence information obtained from the analysis of
7. Basic steps involved in the generation of cDNA from an mRNA template. A cDNA strand is generated with the enzyme reverse transcriptase, with a synthetic oligo-dT primer that attaches to the poly(A) tail of mRNA to initiate DNA synthesis. The RNA is degraded with alkali, and a second DNA strand is generated with the first strand as a template. Additional steps required for the processing of the DNA product are not illustrated.
Figure
Table 1. Practical
applications
Figure 8. Protein synthesis mechanism, illustrating the generation of a correct amino acid sequence by matching triplet codons with complementary sequences in amino acid-specific transfer RNA (tRNA) species.
of cDNA with relevance to nutrition
11
cDNA clones has contributed significantly to our knowledge of the structure, function, and regulation of each of these proteins, as well as their roles in human disease states. Northern Blotting. Once a cDNA clone has been obtained, it can be used as a probe to assess the level of expression of its corresponding mRNA in cells or tissues. This is most often accomplished by the method of Northern blotting, which is outlined in Figure 9. RNA is isolated from a tissue of interest and run through a gel matrix in an electrical field that spreads out or separates the different RNA species according to their sizes (ie, number of nucleotides). The RNA is then transferred from the gel to a paperlike material, usually nitrocellulose, by a process called blotting. The cDNA or a fragment of the cDNA corresponding to an mRNA of interest is made into a cDNA probe by radioactive labeling (usually with 32P)
and is allowed to interact with the blot. It attaches specifically to mRNA with complementary sequence through the process of base pairing. A piece of photographic film can then be placed over the blot, and the radiation released from the cDNA probe exposes the film. By this procedure, the location of a specific mRNA species on the blot (and thus its size) can be established and the amount of mRNA present can be determined from the density of the exposed region on the film. Figure 10 illustrates the use of the Northern blotting method to study the expression of mRNA for IGF-I in fetal liver during maternal fasting in the rat.13 Several IGF-I mRNA transcripts of different sizes are evident in the Northern blot of liver RNA from six control animals (shown in the upper panel of the figure). All of these mRNA species are markedly decreased in quantity in the livers of fetuses obtained from fasted pregnant rats (shown in the lower panel).
Table 2. Nutritionally relevant protein sequences derived from cDNA
Figure 9. Basic principles of Northern blotting. RNA is isolated from a tissue or cell of interest, separated on the basis of size by gel electrophoresis, and transferred to nitrocellulose paper. After allowing a labeled cDNA probe to hybridize to mRNA with a complementary nucleotide sequence, the specific mRNA species can be identified as individual bands by autoradiographically exposed film. The size of the mRNA can be determined by comparison with standards of known size, and the amount is proportional to the density of the band.
12
correctly translated by bacterial riboAlthough conceptually simple, the creation of protein-producing bacterial factories is a complex technology that has required: the construction of specialized plasmids called expression vectors, which produce a cDNA sequence readily translated into protein by bacteria, the use of bacterial strains optimized for protein production, and the application of innovative biochemical methods to finally obtain a protein that is close enough in structure to the human protein sequence
were
somes.
Figure 10. Northern blot illustrating the effects of maternal fasting on IGF-I mRNA in fetal rat liver. The multiple bands in each lane represent different-sized IGF-I mRNA species. Each numbered lane contains RNA from a different animal. Reproduced from Straus et aF3 with permission. kb, kilobase.
This hormone is an important determinant of tissue growth rates, and its regulation during states of nutritional deprivation is likely to be an important factor in determining fetal growth as well as associated congenital malformations. The Northern blotting technique can also be applied to human nutritional studies with RNA preparations obtained, for example, from needle biopsies of skeletal muscle.&dquo; Analysis of levels of mRNA coding for specific muscle proteins, such as myosin, can provide insight into the factors that influence protein synthetic rates and overall tissue nitrogen balance. Recombinant Protein Products from Bacteria. The power of cDNA technology is dependent in large part on the availability of relatively simple methods to greatly amplify the number of copies of a specific cDNA sequence once it has been cloned. As was illustrated in Figure 6, this can be accomplished simply by infecting a culture of bacteria with a plasmid that contains the cDNA of interest and then allowing the plasmid to replicate to high levels within the bacteria. The host bacteria become cDNA-producing biological factories. Because the cDNA contains a sequence that is equivalent to the nucleotide sequence of mRNA, large quantities of the protein encoded by the cDNA could be synthesized by the bacteria if the cDNA
of interest to be useful. The economic incentives and the potential clinical importance of producing large quantities of human proteins through these recombinant DNA methods have stimulated an enormous amount of research and have led to considerable success in this area in the past several years. Table 3 presents a partial listing of proteins with importance in the field of nutrition that have been produced by this application of recombinant DNA1 technology. The use of human recombinant insulin has largely replaced the use of pork and beef insulin for the treatment of diabetes mellitus in many parts of the world and has averted a potential crisis as the need for insulin threatened to exceed available supplies from slaughterhouse-derived animal tissues.&dquo; Human recombinant growth hormone’ became available for clinical use in children with growth hormone deficiency at about the time that human pituitaryderived growth hormone was recognized to be hazardous because of potential pathological viral contaminants. 16 Recent studies have suggested that growth hormone may have therapeutic usefulness as an anabolic agent in nutritionally depleted patients. 17 Other hormones, such as IGF-I18 and granulocyte-macrophage colony stimulating factor,19 may also have important clinical applications. The recognition that trophic hormones and nutrients may have additive anabolic effects or may even act synergistically2° has generated a considerable amount of interest among nutrition researchers. A new era in the field of nutritional support may develop in which the specific composition and quantity of nutrients provided to many catabolic, critically ill patients are determined, in part, by the coordinate administration of recombinant, tissue-specific trophic hormones.
Recombinant Protein Expression in Cultured Cells. By using methods conceptually similar to those that Table 3. Nutritionally relevant recombinant DNA technology
proteins produced by
13
have been used to introduce recombinant DNA into bacteria and to induce the production of specific cloned proteins, it is possible to modify mammalian cells in culture such that they also produce large quantities of proteins encoded by cDNA clones.21 The introduction of foreign cDNA into mammalian cells, through the process called transfection, provides a powerful tool for studying the function of both normal and abnormal proteins. Proteins involved in nutritionally important pathways, such as insulin receptors,22 IGF receptors,23 or low-density lipoprotein receptors,24 can be transfected into cultured cells, and the consequences of their expression at high levels on the cell surface can be studied to gain insight into their normal effects on cellular metabolism. By modifying the sequence of the transfected cDNA by methods of sitedirected mutagenesis,25 the structures of the expressed proteins can be changed and the specific relationships between protein structure and function can be defined.26 Naturally occurring mutant cDNA sequences isolated from patients with inherited metabolic disorders can similarly be transfected into cultured cells, and the consequences of the mutation for the function of the protein can be investigated. By this method, it has recently been possible to demonstrate the molecular basis for certain rare forms of diabetes and growth disorders.&dquo; As the genetic bases for inherited propensities to many nutritionally related disorders, including various forms of obesity, lipid disorders, and digestive disorders, are identified in the years ahead, studies in transfected cultured cells will provide a valuable tool for establishing the functional properties of the abnormal or modified proteins and, thus, the basic molecular events that result in clinical disease. Gene and Protein Expression in Intact Organisms. An exciting recent application of transfection techniques is their use to introduce cDNA into intact animals. An animal that has received foreign DNA in this manner is referred to as transgenic.28 This is an area of intense research interest, because it offers the potential not only to investigate protein functions in vivo in a manner analogous to that described for transfected cultured cells, but it also has possible therapeutic applications as one form of corrective gene therapy. Most work has thus far been carried out in mice, and the method most commonly employed at present involves the injection of a specialized plasmid containing the cDNA of interest into a fertilized mouse egg under direct vision (Fig 11). Several eggs are then implanted into a hormonally primed female mouse, and the progeny are examined for the presence of the injected DNA sequence and phenotypic or biochemical consequences of the expression of the protein encoded by the injected DNA sequence. Under favorable conditions, the injected DNA is stably incorporated into the mouse genome and thus becomes an inherited trait that can be studied in the progeny of the initial transgenic mouse. The result can be dra-
11. Microinjection of fertilized mouse egg with foreign DNA. The egg is immobilized on a pipet by gentle suction. Reproduced from Gordon and Ruddle29 with permission.
Figure
matic, as illustrated in the transgenic mouse in Figure 12 expressing high levels of an injected growth hormone
gene.3° This methodology has obvious important
implications for studying protein functions in vivo and for correcting genetic abnormalities. It also has potential applications as a method for modifying the quality and composition of natural nutritional products obtained from plant and animal sources. It has become near-future science fiction to envision &dquo;transgenic supercattle&dquo; producing low-fat milk, low-cholesterol beef, or even human milk.31 SUMMARY AND A LOOK TO THE FUTURE
In the past several years, there has been a flourishing of new technologies as the science of molecular biology has rapidly developed. It has been possible in this review to discuss briefly only a few of the methods and experimental approaches that have current or potential application in the field of nutrition. Some of the applications of this technology that can be expected in the near future are summarized in Table 4. The cloning of cDNA corresponding to enzymes, transport proteins, hormones, plasma proteins, and other diverse proteins that have nutritionally relevant functions can be anticipated. By methods similar to those described in this review, the study of these proteins will yield new insights into molecular regulatory mechanisms and will also lead to the development of new recombinant DNA products. By powerful new methods for studying the genetic basis of disease at the molecular level, the specific genetic determinants of disease and risk factors for diseases such as obesity, atherosclerosis, neoplasia, and classical inborn errors of metabolism will be defined at an even more rapid pace. Methods are being developed for altering these genetic factors through gene modification or various forms of gene replacement. New disciplines may develop within the field of nutrition that are defined by this new knowledge. For example, as
14 REFERENCES 1. Villa-Komaroff L, Efstratiadis A, Broome S, et al. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci. USA 1978;
75:3727-31. 2. Martial
3.
4.
5. 6. 7. 8.
9.
Figure
12. Two
sibling male
mice at approximately 10 weeks of age. The animal on the left (44 g) contains the rat growth hormone gene fused to the mouse metallothionein promoter. The animal on the right (29 g) is a normal control. Photograph courtesy of Ralph Brinster.
Table 4. Future in nutrition
10.
11.
applications of molecular biology
JA, Hallewell RA, Baxter JD, et al. Human growth hormone: complementary DNA. Cloning and expression in bacteria. Science 1979;205:602-7. Buell G, Schulz MF, Selzer G, et al. Optimizing the expression of a synthetic gene encoding somatomedin-C (IGF-I). Nucleic Acids Res 1985;13:1923-38. Kunkel LM, Hoffman EP. Duchenne/Becker muscular dystrophy : a short overview of the gene, the protein; and current diagnostics. Br Med Bull 1989;45:630-43. McPherson MA, Dormer RL. Molecular and cellular biology of cystic fibrosis. Mol Aspects Med 1991;12:1-81. Allende JE. The human genome initiative. FASEB J 1991;5: 6-78. Lewin B. Genes IV. Cambridge: Cell Press, 1990. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989. Wernerman J, von der Decken A, Vinnars E. Size distribution of ribosomes in biopsy specimens of human skeletal muscle during starvation. Metabolism 1985;34:665-9. Wernerman J, von der Decken A, Vinnars E. The interpretation of ribosome determination to assess protein synthesis in human skeletal muscle. Infusionstherapie 1986;13:162-5. Hammarqvist F, Wernerman J, Ali R, et al. Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis, and improves nitrogen balance. Ann
Surg 1989;209:455-61. RJ, Wilmore DW. Glutamine nutrition and require1990;14:94S-9S. 13. Straus DS, Ooi GT, Orlowski CC, et al. Expression of the genes for insulin-like growth factor-I (IGF-I), IGF-II, and IGF-bind-
12. Smith
ments. JPEN
ing proteins-1 and -2 in fetal rat under conditions of intrauterine growth retardation caused by maternal fasting. Endocrinol14.
15.
ogy 1991;128:518-25. Fong Y, Minei JP, Marano MA, et al. Skeletal muscle amino acid and myofibrillar protein mRNA response to thermal injury and infection. Am J Physiol 1991;261:R536-42. Brogden RN, Heel RC. Human insulin. A review of its biological activity, pharmacokinetics and therapeutic use. Drugs 1987;34:
350-71. 16. Tintner
of the regulation of the expression of specific genes advances, there may be opportunities for directed nutrient regulation of target genes as a component of practical therapy. It should be apparent to the reader that molecular biology can be viewed as a set of new tools available to basic and clinical researchers. To be effectively used, this methodology must ultimately be integrated with other basic sciences, such as biochemistry, metabolism, and physiology, as well as with the clinical disciplines that directly confront the problems presented by human disease. The challenge for health care workers in clinical nutrition is to understand the new technology and to actively participate in its Ma effective application to clinical practice.
knowledge
m
17.
18.
19.
20.
21.
R, Brown P, Hedley-Whyte ET, et al. Neuropathologic
verification of Creutzfeldt-Jakob disease in the exhumed American recipient of human pituitary growth hormone: epidemiologic and pathogenetic implications. Neurology 1986;36:932-6. Manson JM, Smith RJ, Wilmore DW. Growth hormone stimulates protein synthesis during hypocaloric parenteral nutrition. Role of hormonal-substrate environment. Ann Surg 1988; 208:136-42. Skottner A, Clark RG, Fryklund L, et al. Growth responses in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor I. Endocrinology 1989;124: 2519-26. Crawford J, Ozer H, Stoller R, et al. Reduction by granulocyte colony-stimulating factor of fever and neutropenia induced by chemotherapy in patients with small-cell lung cancer. N Engl J Med 1991;325:164-70. Jacobs DO, Evans DA, Mealy K, et al. Combined effects of glutamine and epidermal growth factor on the rat intestine.
Surgery 1988;104:358-64. Scangos G, Ruddle FH. Mechanisms and applications of DNA mediated gene transfer in mammalian cells; a review. Gene 1981;14:1-10.
ACKNOWLEDGMENT
The author thanks Drs. Michael Pedrini and Thomas R. Ziegler for careful reading of the manu-
script.
22. Ebina Y, Edery M, Ellis L, et al. Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells. Proc Natl Acad Sci USA 1985;82:8014-8. 23. Steele-Perkins G, Turner J, Edman JC, et al. Expression and characterization of a functional human insulin-like growth factor I receptor. J Biol Chem 1988;263:11486-92.
15 24.
Miyanohara A, Sharkey MF, Witztum JL, et al. Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit
fibroblasts in vitro. Proc Natl Acad Sci USA 1988;85:6538-42. 25. Harris T. In vitro mutagenesis. Nature 1982;299:298-9. 26. Ellis L, Clauser E, Morgan DO. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell 1986; 45:721-32. 27. Moller DE, Flier JS. Mechanisms of disease. Insulin resistance—mechanisms, syndromes, and implications. N Engl J
Med 1991;325:938-48. RD, Brinster RL. Germ-line transformation of mice. Annu Rev Genet 1986;20:465-99. 29. Gordon JW, Ruddle FH. Gene transfer into mouse embryos: production of transgenic mice by pronuclear injection. Methods
28. Palmiter
Enzymol 1982;101:411-33. 30. Palmiter RD, Brinster RL, Hammer RE, et al. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 1982;300:611-5. 31. Johnson J. Transgenics: the land of milk and money. J NIH Res 1991;3:26-7.
